CN102205132B - Preparation formula of gene therapy medicament taking recombinant adeno-associated virus (rAAV) as vector - Google Patents
Preparation formula of gene therapy medicament taking recombinant adeno-associated virus (rAAV) as vector Download PDFInfo
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- CN102205132B CN102205132B CN201110123404.6A CN201110123404A CN102205132B CN 102205132 B CN102205132 B CN 102205132B CN 201110123404 A CN201110123404 A CN 201110123404A CN 102205132 B CN102205132 B CN 102205132B
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Abstract
The invention discloses a preparation formula of a gene therapy medicament taking a recombinant adeno-associated virus (rAAV) as a vector. A preparation in the formula comprises a rAAV gene therapy medicament as well as excipient components such as a protective human serum albumin for preventing container adsorption, a buffer pair of citric acid and sodium citrate for maintaining the pH value of a solution in a neutral range and a surfactant polyether F-68 for preventing polymerization among rAAV gene vectors. The preparation does not cause the rapid descending of the biological activity under a room temperature condition, and does not require cryopreservation.
Description
Technical field
The present invention relates to a kind of preparation prescription of take the gene therapy medicament that recombinant adeno-associated virus is carrier, belong to molecular medicine, molecular medicine and diseases prevention and treatment field.
Background technology
The disease that gene therapy causes for therapeutic gene defect provides novel solution, the gene therapy of several major diseases succeeds recently, indicate that gene therapy has obtained important breakthrough, thereby by Science magazine one of ten big sciences progress [the Alberts B.Science that is chosen as 2009,2009,326 (5960): p1589].Successful story just comprises applies the clinical research that recombinant adeno-associated virus (rAAV) genomic medicine is successfully cured Leber ' s congenital amaurosis cataract.The galenic pharmacy research of the genomic medicine based on viral vector is a relatively new field, seldom sees so far some systematic researches to solve preparation prescription optimization and the stability problem of viral vector genomic medicine.Although can obtain inspiration from the prescription of vaccine and recombinant protein product, but the genomic medicine based on viral vector has essential difference with vaccine and recombinant protein medicine after all, existing result of study also shows that the preparation prescription of viral vector genomic medicine and stability are the new challenges that preparation research personnel face.
For a long time, everybody unanimously think rAAV genomic medicine have no pathogenicity, reduced immunogenicity, can mediate foreign gene long-term expression and the advantage such as host range is extensive, be the most promising gene therapy vector.Our early-stage Study shows, purified rAAV genomic medicine stores immediately under-80 ℃ of conditions, and within 3 months, its inside and outside transduction efficiency [is permitted Ruian .Med Sci Monit, 2005 without significant change; 11:BR305-308].But it is harmless that rAAV genomic medicine must be replaced by before human body application, and can under cold preservation or room temperature condition, stablize the solvent of depositing and facilitating transportation.By the solvent replacement of rAAV genomic medicine, be under-80 ℃ or-20 ℃ of conditions, to store after the phosphate buffer that laboratory is conventional (PBS, pH 7.0), the titre of once transduceing of often thawing all declines more than 20%; Under 4 ℃ of conditions, store one day transduction titre 10% left and right that declines.So the selection of suitable solvent is to guarantee rAAV genomic medicine key of preserving steady in a long-term.
Reducing to greatest extent rAAV genomic medicine in storage process significant biochemical variation occurs, guarantee that rAAV genomic medicine has enough biologic activity when human body is applied, is the significant challenge that its galenic pharmacy exploitation faces.Storage requirement must be obeyed the market-oriented requirement of medicine, and as transportation and storage stage do not need harsh freezing preservation, the room temperature condition before patient's use can not cause bioactive rapid lower degradation.
Summary of the invention
For addressing the above problem, the object of the present invention is to provide a kind of preparation prescription of take the gene therapy medicament that recombinant adeno-associated virus is carrier, said preparation can not cause bioactive rapid decline at room temperature condition, and does not need freezing preservation.
In order to achieve the above object, technical scheme of the present invention is:
A kind of preparation prescription of take gene (rAAV) medicine that recombinant adeno-associated virus is carrier, in said preparation except containing rAAV gene therapy medicament (being purchased from U.S. University of Pennsylvania), also add following excipient component: the protective agent human albumin (being purchased from Shanghai Laishi Blood Product Co., Ltd) who prevents container adsorption, maintain pH value of solution in the buffering of neutral range to citric acid and sodium citrate (being purchased from Nanjing chemical reagent factory), prevent from occurring between rAAV genophore the surfactant polyethers F-68 (P] uronic F-68) (being purchased from BASF Aktiengesellschaft) of polymerization.
The genome titre (vg) of described medicine is 10
8-10
14vg.
Described preparation formulation is a kind of in injection, freeze-dried powder or solution.
Described preparation is injection or solution, and when molten coal is pure water or water for injection, in prescription, add the amount of excipient component to be: in every 1ml water for injection, to add human albumin 0.5-5mg, sodium citrate 10-50mg, citric acid 0.1-1.0mg, polyethers F-68 0.001-0.1mg.
In described prescription, preferably add the amount of excipient component to be: in every 1ml water for injection, to add human albumin 2.5mg, sodium citrate 30mg, citric acid 0.6mg, polyethers F-680.001mg.
When described preparation is freeze-dried powder, in prescription, add the amount of excipient component to be: in every preparation unit (being every injection), to add human albumin 0.5-5mg, sodium citrate 10-50mg, citric acid 0.1-1.0mg, polyethers F-68 0.001-0.1mg, mannitol 50-500mg.
In described prescription, preferably add the amount of excipient component to be: in per unit preparation, to add human albumin 2.5mg, sodium citrate 30mg, citric acid 0.6mg, polyethers F-68 0.001mg, mannitol 100mg.
Beneficial effect of the present invention is: the present invention screen and the rAAV genomic medicine injection prescription optimized in contain protective agent human albumin, buffering is to citric acid and sodium citrate, surfactant polyethers F-68.In the prescription of freeze-dried powder, except containing mentioned component, also contain mannitol.Said preparation prescription has solved rAAV genomic medicine bioactive object of long term maintenance under stored refrigerated condition, can not cause bioactive rapid decline, and do not need freezing preservation at room temperature condition.The rAAV genomic medicine of applying this prescription stores at refrigerated condition (2-8 ℃), and effect duration is 1 year.Room temperature (10-30 ℃) is preserved 1 day, and biologic activity declines and is no more than 10%.Can be applicable to the clinical use product of rAAV genomic medicine, for clinical research and the market development of the genomic medicine based on rAAV carrier provides guarantee.
Accompanying drawing explanation
Fig. 1 is that the present invention is melted artifact at-80 ℃ of frozen rAAV genomic medicines again and learned active situation of change.Human albumin, buffering is to citric acid and sodium citrate, and adding of surfactant polyethers F-68 can significantly reduce the destruction of frozen-thaw process to biologic activity.
Fig. 2 is that the present invention is melted artifact at-20 ℃ of frozen rAAV genomic medicines again and learned active situation of change.Human albumin, buffering is to citric acid and sodium citrate, and adding of surfactant polyethers F-68 can significantly reduce the destruction of frozen-thaw process to biologic activity.
Fig. 3 is the situation of change of the rAAV genomic medicine biologic activity of the present invention under 4 ℃ of preservations.Human albumin, buffering is to citric acid and sodium citrate, and surfactant polyethers F-68 adds the reduction that can significantly reduce biologic activity in storage process.
Fig. 4 is the situation of change of the rAAV genomic medicine biologic activity of the present invention under room temperature preservation.
The specific embodiment
Embodiment 1
The preparation of rAAV genomic medicine stock solution
For ease of measuring the transduction titre (biologic activity) of rAAV genomic medicine, the transgenic of the present embodiment medicine is LUC Photinus pyralis LUC Photinus pyralis FL (Fluc) gene (being purchased from Promega company).
The preparation method of the rAAV gene therapy vector that the present embodiment is used is: the many plasmid co-transfections legal system that adopts non-auxiliary virus to participate in is standby, adopt calcium phosphate method by vector plasmid and helper plasmid (as H22, H23, H24, H25, H26, H27, H28, H29, pXX6 etc.) (being purchased from U.S. University of Pennsylvania) cotransfection 293 cells (be purchased from US mode strain and collect center (ATCC)), after 60-72 hour, gather in the crops recombinant virus, standby after caesium chloride density gradient centrifugation purification.Described helper plasmid adopts AAV helper plasmid (H22,, H23, H24, H25, H26, H27, H28, H29) and adenovirus helper plasmid (pXX6) all purchased from U.S. University of Pennsylvania.Concrete steps are: 1. in disposable 50-ml polystyrene tube, mix following material: 90 μ g pXX6 helper plasmids, 30 μ g rAAV vector plasmids, 30 μ g H22 helper plasmids, 0.4ml 2.5M CaCl
2, add deionized purified water to 4ml.
2. add in the mixture that 1. 4ml 2 * HEPES buffer salt solution obtain in step, turn upside down 3 times and mix, at incubated at room 1-5 minute.
3. in 5 seconds of mixture that 2. vortex oscillation step obtains, dropwise add on the cell flat board of cultivating the 15cm that has 293 cells, and every plate drips 2mL.Rotating Plates makes Monodispersed.
4. 293 cells after three plasmid co-transfections continue to cultivate results after 60-72 hour.Freeze-thaw method smudge cells, through ammonium sulfate precipitation, the conventional method purification such as caesium chloride density gradient centrifugation, obtain the stock solution rAAV2/Fluc of rAAV genomic medicine, and-80 ℃ store for future use.
H22 helper plasmid is replaced by H21, H23, H24, H25, H26, H27, H28, H29, stock solution rAAV1/Fluc, rAAV3/Fluc, rAAV4/Fluc, rAAV5/Fluc, rAAV6/Fluc, rAAV7/Fluc, rAAV8/Fluc, rAAV9/Fluc with legal system for different serotypes rAAV genomic medicine ,-80 ℃ store for future use.
Embodiment 2
RAAV genomic medicine transduction titre (biologic activity) is measured
1. get 293 cells and cultivate at 6 porocyte culture plates, cell density is 10
5/ hole.
2. get rAAV genomic medicine through suitably dilution, add in step Tissue Culture Plate 1., the addition of every hole rAAV genomic medicine is 10
6genome titre (vg).
3. adopt the transduction titre of the luciferase reporter gene detection kit mensuration rAAV genomic medicine rAAV/Fluc of Bi Yuntianchang production, with expression (pg) calculating of Fluc in every hole.
Embodiment 3
The stock solution of getting the rAAV genomic medicine of preparation in embodiment 1, through ultrafiltration replacement solvent, obtains respectively the preparation in table 1.It is control solvent that the laboratory of take is commonly used PBS buffer (pH 7.4).Table 1 is the preparation table of rAAV genomic medicine stability study preparation, the wherein preparation of PBS buffer: Na
2hPO
412H
2o 3.868mg, NaH
2pO
42H
2o 0.395mg, NaCl6.8mg, is dissolved in 1ml water for injection.Certainly in prescription, add the amount of excipient component all can realize object of the present invention for the arbitrary value in following scope: in every 1ml water for injection, to add human albumin 0.5-5mg, sodium citrate 10-50mg, citric acid 0.1-1.0mg, polyethers F-68 0.001-0.1mg.
Embodiment 4
Get the preparation that embodiment 3 obtains, respectively through 0.2 μ m filter aseptic filtration 2 times.According to method described in embodiment 2, measure transduction titre.The results are shown in Table 2, table 2 is the bioactive loss table of aseptic filtration process rAAV genomic medicine.By table 2, can be obtained: take PBS as solvent, rAAV genomic medicine just obviously bioactive loss has occurred in the process of aseptic filtration, and particularly when low concentration, downward trend is more obvious.But can reduce the trend that its biological activity loses after adding albumin and polyethers F-68.
Citric acid buffer system also helps avoid the loss of activity of rAAV genomic medicine in the process of aseptic filtration.No. 3-1 prescription is better than prescription No. 3-2.
Embodiment 5
Get the preparation that embodiment 3 obtains ,-80 ℃, warp is freezing with-20 ℃ respectively, after thawing, according to method described in embodiment 2, measures transduction titre.The results are shown in Figure 1 and Fig. 2.Fig. 1 is that-80 ℃ of refrigerating process are on the active figure that affects; Fig. 2 is that-20 ℃ of refrigerating process are on the active figure that affects; As seen from the figure, the rAAV genomic medicine that the PBS of take is solvent is through after-80 ℃ and-20 ℃ of cold melting, and biologic activity reduces and surpasses 20%.After adding, albumin and polyethers F-68 can reduce the biological activity loss that rAAV genomic medicine causes because of freezing thawing.3-1 and 3-2 preparation better effects if, freezing thawing is once almost without the variation of obvious biological activity.
Embodiment 6
Get the preparation that embodiment 3 obtains, respectively 4 ℃ and room temperature placement, according to method described in embodiment 2, measure the transduction titre of different time.The results are shown in Figure 3 and Fig. 4.Fig. 3 is 4 ℃ of preservation condition stability inferior result figure; Fig. 4 is room temperature preservation condition stability inferior result figure.As seen from the figure, under 4 ℃ of preservation conditions, the biologic activity of the rAAV genomic medicine that the PBS of take is solvent reduced by 50% left and right in 1 month.After adding, albumin and polyethers F-68 can reduce its biologic activity downward trend.3-1 and 3-2 preparation biologic activity in 1 year can maintain more than 90%.
When room temperature is placed, 3-1 preparation biologic activity can maintain more than 90% in 2 days, and 3-2 preparation can maintain more than 90% in 1 day, and biologic activity declines day by day afterwards.The rAAV genomic medicine of the prompting side of Clicking here preparation, from refrigerated condition takes out, can be placed 1 day at room temperature condition, has facilitated patient's application.Minute packaging process that simultaneously also prompting completes rAAV genomic medicine at ambient temperature in 1 day on stability of drug products without impact.
Embodiment 7
Get rAAV1/Fluc, rAAV3/F that embodiment 1 obtains] uc, rAAV4/Fluc, rAAV5/Fluc, rAAV6/Fluc, rAAV7/Fluc, rAAV8/Fluc, rAAV9/Fluc stock solution, according to 3-1 preparation prescription, being mixed with respectively drug content is 10
14the injection of vg/ml, and drug content is 10
9the solution of vg/ml.Refrigerated condition (2-8 ℃) is preserved, and measures the transduction titre of different time according to method described in embodiment 2.Result all samples its transduction titre within the observation period of 1 year all maintains more than 90%, illustrates that 3-1 preparation prescription is all applicable to the rAAV genomic medicine of different serotypes.
Embodiment 8
Get rAAV1/Fluc, rAAV3/F that embodiment 1 obtains] uc, rAAV4/F] uc, rAAV5/Fluc, rAAV6/Fluc, rAAV7/Fluc, rAAV8/Fluc, rAAV9/Fluc stock solution, according to 3-1 preparation prescription, being mixed with respectively drug content is 10
13the injection of vg/ml, and drug content is 10
9the solution of vg/ml.Refrigerated condition (2-8 ℃) is preserved, and measures the transduction titre of different time according to method described in embodiment 2.Result all samples its transduction titre within the observation period of 1 year all maintains more than 90%, illustrates that 3-1 preparation prescription is all applicable to the rAAV genomic medicine of different serotypes.
Embodiment 9
According to 3-1 preparation prescription preparation solvent for injection, in prescription, add in addition mannitol, in every preparation unit (being every injection), contain human albumin 2.5mg, sodium citrate 30mg, citric acid 0.6mg, polyethers F-680.001mg, mannitol 100mg.Certainly in prescription, add the amount of excipient component all can realize object of the present invention for the arbitrary value in following scope: in every preparation unit (being every injection), to add human albumin 0.5-5mg, sodium citrate 10-50mg, citric acid 0.1-1.0mg, polyethers F-68 0.001-0.1mg, mannitol 50-500mg.
Get rAAV1/Fluc, rAAV2/F that embodiment 1 obtains] uc, rAAV3/Fluc, rAAV4/Fluc, rAAV5/Fluc, rAAV6/Fluc, rAAV7/Fluc, rAAV8/Fluc, rAAV9/F] uc stock solution, being mixed with respectively drug content is 10
14the injection of vg/ml.According to conventional freeze-dry process lyophilization, obtain freeze-dried powder.Room temperature condition is preserved, and measures the transduction titre of different time according to method described in embodiment 2.Result all samples loses biologic activity approximately 50% in freeze-drying process.Biologic activity after completing with lyophilizing is designated as 100%, and within the observation period of 1 year, its transduction titre all maintains more than 90%.Illustrate that freeze-drying process can cause the biologic activity loss of the rAAV genomic medicine of different serotypes, but preparation after lyophilizing can be stablized preservation at ambient temperature.
Table 1
Table 2
| Preparation code name | Transduction titre (pg/ hole) before filtering | Transduction titre (pg/ hole) after filtering | After filtering/before filtering |
| No. 1-1 | 12037±4123 | 10128±3265 | 0.84 |
| No. 1-2 | 10085±4832 | 3875±1681 | 0.38 |
| No. 2-1 | 12392±3696 | 11241±3387 | 0.91 |
| No. 2-2 | 11874±2956 | 7352±2603 | 0.62 |
| No. 3-1 | 12210±3283 | 11965±3281 | 0.98 |
| No. 3-2 | 11957±3624 | 10186±3750 | 0.85 |
Claims (5)
1. take the preparation of the gene therapy medicament that recombinant adeno-associated virus is carrier for one kind, in said preparation except containing rAAV gene therapy medicament, it is characterized in that also adding following excipient component: the protective agent human albumin who prevents container adsorption, maintain pH value of solution in the buffering of neutral range to citric acid and sodium citrate, prevent from occurring between rAAV genophore the surfactant polyethers F-68 of polymerization;
Described preparation formulation is a kind of in injection, freeze-dried powder or solution;
Described preparation is injection or solution, and when solvent is pure water or water for injection, in prescription, add the amount of excipient component to be: in every 1ml water for injection, to add human albumin 0.5-5mg, sodium citrate 10-50 mg, citric acid 0.1-1.0 mg, polyethers F-68 0.001-0.1mg.
2. a kind of preparation of take the gene therapy medicament that recombinant adeno-associated virus is carrier as claimed in claim 1, is characterized in that the genome titre (vg) of described medicine is 10
8-10
14vg.
3. a kind of preparation of take the gene therapy medicament that recombinant adeno-associated virus is carrier as claimed in claim 1, it is characterized in that adding the amount of excipient component to be in described prescription: in every 1ml water for injection, add human albumin 2.5mg, sodium citrate 30 mg, citric acid 0.6 mg, polyethers F-68 0.001 mg.
4. a kind of preparation of take the gene therapy medicament that recombinant adeno-associated virus is carrier as claimed in claim 1, while it is characterized in that described preparation is freeze-dried powder, in prescription, add the amount of excipient component to be: in every preparation unit, to add human albumin 0.5-5mg, sodium citrate 10-50 mg, citric acid 0.1-1.0 mg, polyethers F-68 0.001-0.1mg, mannitol 50-500 mg.
5. a kind of preparation of take the gene therapy medicament that recombinant adeno-associated virus is carrier as claimed in claim 4, it is characterized in that adding the amount of excipient component to be in described prescription: in every preparation unit, add human albumin 2.5mg, sodium citrate 30 mg, citric acid 0.6 mg, polyethers F-68 0.001 mg, mannitol 100mg.
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| KR102069561B1 (en) * | 2013-07-11 | 2020-01-23 | 다카라 바이오 가부시키가이샤 | Method for manufacturing non-enveloped virus |
| US10023846B2 (en) | 2014-07-10 | 2018-07-17 | Takara Bio Inc. | Production method for non-enveloped virus particles |
| CN107109374B (en) | 2015-01-09 | 2021-04-06 | 宝生物工程株式会社 | Method for producing non-enveloped virus particles |
| KR102167829B1 (en) * | 2020-02-10 | 2020-10-20 | 주식회사 이노테라피 | Stabilizers for adeno-associated viruses and methods for stabilizing adeno-associated viruses using the Same |
| CN114621393A (en) * | 2022-03-17 | 2022-06-14 | 华侨大学 | Copolymer of N- (2-hydroxy) propyl methacrylamide and N-methacryloyl glycyl glycine and application thereof |
| CN115337408A (en) * | 2022-09-16 | 2022-11-15 | 上海信致医药科技有限公司 | Adeno-associated virus formulations |
| CN115554418B (en) * | 2022-11-22 | 2023-04-14 | 四川至善唯新生物科技有限公司 | Pharmaceutical composition of recombinant adeno-associated virus vector and application thereof |
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